CONTROL OF ARG-2 GENE EXPRESSION IN NEUROSPORA

神经孢子虫中 ARG-2 基因表达的控制

• 批准号: 3468809
• 负责人: MATTHEW Steven SACHS
• 金额: $ 15.91万
• 依托单位: OREGON GRADUATE INSTITUTE SCIENCE & TECH
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468809
• 关键词: DNA binding protein; Neurospora; RNA binding protein; alleles; developmental genetics; fusion gene; gel electrophoresis; gene expression; gene mutation; genetic regulatory element; molecular cloning; northern blottings; nucleic acid sequence; reporter genes; transcription factor; translation factor

The long-term goal of this program is to understand the molecular mechanisms by which different regulatory pathways interact to control expression of the Neurospora crassa arg-2 gene. This gene encodes the mitochondrially localized small subunit of arginine-specific carbamoyl phosphate synthetase. The major goal of this work is to define the intragenic and extragenic elements responsible for regulation of Arg-2 by Arg availability. arg-2 is regulated by at least three distinct mechanisms: (1)Arg-specific control negatively regulates expression in response to Arg. (2)Cross-pathway control positively regulates expression in response to limitation for a variety of amino acids, including Arg. (3) Developmental control regulates arg-2 expression in response to as yet unidentified developmental signals. Each of these pathways influences the level of arg-2 transcript. There is evidence for a translational component to Arg-specific control mediated through a 24 codon upstream open reading frame (uORF) in the transcript. Specific aims are as follows: 1. The contributions of transcriptional and translational components to regulation by Arg will be defined. The effects of existing mutations in arg-2 and at regulatory loci will be examined. 2. Novel mutations that affect gene expression in response to Arg will be obtained and characterized. Selection can be accomplished using cells harboring a recombinant gene in which E coli hph, which encodes hygromycin phosphotransferase, has been fused to arg-2 control sequences. These cells are resistant to the antibiotic hygromycin in medium lacking Arg and are sensitive to the antibiotic in medium containing Arg. 3. Mutations will be introduced in vitro into arg-2 regulatory sequences, and their effects on regulation will be analyzed in vivo by analyzing the expression of reporter genes - for example E coli lacZ - to which the sequences are fused. 4. The interactions of factors with arg-2 sequences will be characterized through in vitro DNA and RNA-binding studies as a complementary approach to in vivo studies. An understanding of how arg-2 expression is controlled is of practical as will as fundamental interest because N crassa is related to pathogenic fungi and to fungi used for the production of antibiotics.

该计划的长期目标是了解分子 不同的调节途径相互作用以控制 粗糙脉孢菌arg-2基因的表达。 这个基因编码 精氨酸特异性氨基甲酰基的脑内定位小亚基 磷酸合成酶 这项工作的主要目标是定义 负责调节Arg-2的基因内和基因外元件 通过Arg可用性。arg-2受至少三种不同的 机制：（1）Arg特异性控制负调节表达 对Arg的回应（2）交叉途径控制正调节 表达响应于多种氨基酸的限制， 包括Arg。(3)发育控制调节Arg-2表达 对尚未识别的发育信号的反应。 这一切成功都 途径影响Arg-2转录水平。 有证据 对于转译组分，Arg特异性控制通过 转录物中的24个密码子上游开放阅读框（uORF）。 具体目标如下： 1. 转录和翻译成分对 将定义Arg的调节。 现有突变的影响， 将检测Arg-2和AT调节基因座。 2. 影响基因表达的新突变将对Arg产生反应， 获得并表征。 选择可以使用 含有重组基因的细胞，其中大肠杆菌hph编码 潮霉素磷酸转移酶，已融合到Arg-2控制 序列的 这些细胞对抗生素潮霉素有抗性， 缺乏Arg的培养基，对培养基中的抗生素敏感 含有Arg。 3. 突变将在体外引入arg-2调节基因。 序列，以及它们对调控的影响将通过以下方法在体内分析： 分析报告基因的表达-例如大肠杆菌lacZ - 序列与其融合。 4. 因子与arg-2序列的相互作用将是 通过体外DNA和RNA结合研究表征为 体内研究的补充方法。 了解arg-2的表达是如何被控制的具有实际意义 作为基本利益，因为N crassa与 病原真菌和用于生产抗生素的真菌。